Optical Method for the Detection of Alzheimer&#39;s Disease

ABSTRACT

The present subject matter relates to a non-invasive optical imaging method for monitoring early pathological events specific to Alzheimer&#39;s disease (AD), such as the development, amount and location of amyloid plaques. The ability to monitor such events provides a basis for, among other things, AD diagnosis, prognosis and assessment of potential therapies. In addition, the present subject matter introduces novel methods for treating AD and retinal ailments associated with AD. Aβ-plaque detection in living brains is extremely limited, especially at high resolution; therefore the present invention is based on studies focusing on the eyes as an alternative to brain-derived tissue that can be imaged directly, repetitively and non-invasively.

PRIORITY DATA

This application is a continuation of U.S. application Ser. No.13/119,596, filed Jul. 22, 2011, which is a § 371 national stage entryof PCT Application No. PCT/US2009/057569, filed Sep. 18, 2009, whichclaims priority of U.S. Provisional Application No. 61/098,206, filedSep. 18, 2008, the entire disclosures of which are incorporated hereinby reference.

FIELD OF THE SUBJECT MATTER

The present subject matter relates to methods for noninvasive monitoringof early pathological events specific to Alzheimer's disease, and thusincludes methods and systems for the diagnosis, treatment, prognosis andevaluation of response to treatment of AD.

BACKGROUND OF THE SUBJECT MATTER

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Alzheimer's disease (AD) is a common and devastating age-dependentneurodegenerative disease. AD brain pathology is characterized bytypical accumulation of proteolytic products of the amyloid precursorprotein (APP), amyloid-β peptides (Aβ), which form extracellularaggregates termed Aβ plaques. These plaques are believed to contributeto disrupted cellular activities and communication in the brain, leadingto neurotoxic inflammation and neuronal death [2,3]. Molecular imaging,which allows a non-invasive monitoring of pathological processes inliving subjects, has the potential to enhance detection andunderstanding of disease and drug effectiveness. Accordingly, majorefforts have been invested in developing tools to enable noninvasivedetection of amyloid plaques through the skull of living AD patients andanimal models [4-9]; however, noninvasive monitoring of amyloid plaquesis still clinically challenging and of limited availability at highresolution [10-12]. Optical imaging constitutes a powerful,high-resolution and specific tool for in vivo imaging, as recentlydemonstrated using multiphoton microscopy to image Aβ plaques in vivo inthe mouse brain via a cranial window [13]. The present subject matterposes an alternative and noninvasive approach in humans to image theretina of AD patients by optical modalities, provided that Aβ plaquesdevelop in these patients' retinas and share similar properties withthose in the brain.

APP is widely expressed in the retinal ganglion cells (RGCs), anoutgrowth of the central nervous system (CNS), and is transported to theaxonal plasma membrane and the nerve terminals via the optic nerve [14].Formation of plaques in the retina came recently under investigation,especially in two related neurodegenerative disorders: aged-relatedmacular degeneration (AMD) and glaucoma [50-53]. It was unclear whetherAβ-plaques are found in the retina in early or late stage of ADpatients. Past evidence pointed to the presence of Aβ-plaques in retinasof glaucoma and AMD patients and their rodent models. For example, Aβdeposition in the RGC layer has been reported in glaucoma patients [50,51]. In experimental models of glaucoma, apoptosis of RGCs has beenassociated with the accumulation of Aβ-peptides, and agents targetingtheir formation were shown to exert neuroprotective activity [52]. InAMD patients, Aβ deposits were found in drusen that correlated with thelocation of degenerating photoreceptors and retinal pigment epitheliumcells [53].

In a Drosophila transgenic model of AD, based on the targeted expressionof mutated human APP and presenilin (PS) genes, Aβ immunoreactivity wasfound in the compound eye, and in association with retinal photoreceptordegeneration [15]. A recent study demonstrated Aβ deposits in theretinal nerve fiber layer (NFL) and ganglion cell layer (GCL) in ADtransgenic mice at an advanced stage of the disease (later than 10months of age). The Aβ deposits were further correlated withneurodegeneration of the RGCs and with microglial activation [16].

Despite this encouraging research, there remains a need in the art forsystems and methods for the diagnosis, prognosis and treatment of AD.The present subject matter meets these needs by discovering the presenceof Aβ plaques in retinas of postmortem eyes of AD patients. Using miceexpressing mutated forms of the human APP and PS1 genes (APPswe/PS1dE9,referred to here as AD-Tg mice), the present subject matter alsoprovides evidence disclosing the early formation of Aβ plaques in theretina prior to their manifestation in the brain. Furthermore, thepresent subject matter identifies an immune-based therapy, using a weakagonist of a myelin-derived peptide loaded on dendritic cells [17, 18],effective in reducing Aβ plaques in the mouse brains and retinas ofAD-Tg mice. Finally, the subject matter demonstrated that systemicinjection of curcumin (diferuloylmethane), a natural compound that bindsand labels Aβ plaques [19, 20], into live animals allows fornon-invasive high-resolution and specific visualization of Aβ plaques inthe retina. The present subject matter teaches methods that, for thefirst time, allow for Aβ plaques to be detected by number and location,and be repeatedly counted and monitored in real-time in the retina of ADmammals.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein areconsidered illustrative rather than restrictive.

FIGS. 1a through 1n depict retinal Aβ deposition in the retina of AD-Tgmice visualized by curcumin. FIGS. 1a-1f depict images of braincryosections from 9-month-old AD-Tg (FIGS. 1a-1e ) and non-Tg (wt) (FIG.1f ) mice stained with anti-human Aβ antibody and curcumin ex vivo,indicating co-localization of Aβ plaque staining by both detectionmethods. FIGS. 1d and 1e depict higher magnification images of plaquestaining pattern presented for each channel FIG. 1f shows no evidencefor double-positive anti-human Aβplaques and curcumin in the non-Tg (wt)mouse. Cell nuclei were labeled with DAPI (blue). Scale bar=100 μm.FIGS. 1g-1j are representative images of retinal whole-mounts from10-month-old AD-Tg (n=27) and non-Tg (wt) mice (n=18) stained withanti-Aβ antibody and curcumin ex vivo. The formation of Aβ plaques(yellow spots of overlapping red and green channels) is demonstrated inseveral different retinal layers: FIG. 1g depicts IPL-Inner PlexiformLayer, FIG. 1h depicts INL-Inner Nuclear/OPL-Outer Plexiform Layers, andFIG. 1i depicts ONL-Outer Nuclear Layer. FIG. 1j shows AP plaques wereessentially absent in the non-Tg (wt) mice. (FIGS. 1g and 1j , lowerrow). Higher magnification images for separate channels demonstrateplaque-staining patterns with both procedures. Scale bars=5 μm. FIGS.1k-1n depict whole eye sagittal cryosections stained with curcumin invivo, followed by anti-human Aβ antibody and DAPI ex vivo. In FIGS.1k-1m , Aβ plaques were detected in most retinal layers and in thechoroid in 10-month-old AD-Tg mice. In FIG. 1n , Aβ plaques wereundetectable in the retina and choroid of non-Tg (wt) mice. Scale bar=20μm.

FIGS. 2a through 2n depict Aβ plaques in the human retina of Alzheimer'sdisease patients. FIGS. 2a and 2b are representative images of the humanwhole-mount retina of an 87-yr-old AD patient after staining with SudanBlack B to eliminate non-specific autofluorescence signals, andfollowing curcumin ex vivo staining (curcumin-labeled plaques areindicated by white arrows). Scale bars=10 μm. Cell nuclei are labeledwith DAPI (blue). FIGS. 2c and 2d show higher magnification images ofthe human whole-mount retina of a 65-yr-old AD patient following SudanBlack B staining (black spots for Sudan staining), and then curcuminstaining (curcumin-labeled plaque is indicated by white arrow). Scalebars=5 FIGS. 2e-2g provide additional examples of curcumin-positiveplaques in retinas of a series of 65- to 90-yr-old human AD patients.FIGS. 2h-2j are representative images of human whole-mount retinas of65- and 87-yr-old human AD patients stained with anti-human Aβantibodies followed by Sudan Black B treatment at several retinal depths(to include RGC and IPL). FIG. 2i represents a higher magnificationimage of the retinal plaque. Aβ plaque morphology was similar to thatfound in the mouse retinas and brains. Scale bars=5 μm. FIGS. 2k-2mrepresent subsequent staining of the same human retinas with curcumin,which reveals that the plaques were selectively colabeled with human Aβantibodies and curcumin (lower row images for separate channels). FIG.2n depicts double staining with human Aβ antibodies and curcumin inpostmortem non-AD human retinal whole mount showing no signs of Aβplaques (lower row images for separate channels). Scale bars=5 μm.

FIGS. 3a through 3q depict mouse retinal Aβ plaque formations at thepre-symptomatic early stage and accumulation during disease progression.Following i.v. curcumin injections into the tail vein, Aβ plaques werevisible in AD-Tg mice retinas and brains. FIGS. 3a-3n are representativez-axis projection images of whole-mount retinas from AD-Tg (n=18) andnon-Tg (wt; n=10) mice at various ages; FIGS. 3a-3d depict 2.5-month-oldAD-Tg mouse, with FIG. 3a showing presence of plaques in the retina andFIG. 3b showing validation of Aβ plaque staining using specificanti-human antibody ex vivo at the same location (co-localization ofcurcumin and Aβ antibody in yellow). Scale bars=10 μm. FIGS. 3c and 3dshow no plaques were detected in the brain hippocampus and cortex. Scalebars=100 μm. FIGS. 3e-3h depict 5-month-old AD-Tg mouse, with FIG. 3edepicting the presence of plaques in the retina and FIG. 3f followingspecific Aβ antibody staining ex vivo. Scale bars=10 μm. FIGS. 3g and 3hshow detection of plaques in the brain. Scale bars=50 μm. FIGS. 3i-3kdepict 9-month-old AD-Tg mouse, with FIG. 3i showing multiple plaques inthe retina and FIGS. 3j and 3k showing plaques in the brain. Scale bars(i)=10 μm and (j,k)=50 μm. FIGS. 3l-3n depict 17-month-old AD-Tg mouse,with FIG. 3l showing numerous plaques in the retina and FIGS. 3m and 3nshowing plaques in the brain. Scale bars (i)=10 μm and (m,n)=100 μm.FIGS. 3o-3q depicts 9-month-old non-Tg (wt) mouse, with FIG. 3o showingno plaques in the retina and FIGS. 3p and 3q showing no plaques in thebrain. Scale bars (o)=10 μm and (p,q)=100 μm.

FIGS. 4a through 4k depict decreased Aβ plaques in the retina of AD-Tgmice following dendritic cell-based vaccination. FIGS. 4a-4g arerepresentative z-axis projection images of whole-mount retinas from 10month-old mice, FIGS. 4a-4c show PBS-treated AD-Tg mouse control, FIGS.4d-4f show vaccinated AD-Tg mouse, and FIG. 4g shows non-Tg (wt) mousestained ex vivo with curcumin and anti-human Aβ antibodies. FIGS. 4b and4c , and FIGS. 4c and 4f depict separate channel images for curcumin andanti-Aβ antibodies labeling in the retina. Scale bars=10 FIG. 4h is anillustration of 12 regions around the optic disc (indicated byrectangles 1-12) representing the area covered for quantitative analysesof plaques in the retinal whole-mounts (n=4 mice per group; two retinasper mouse). Scale bar=200 FIG. 4i depicts the decrease in plaque number,observed in the retinas of AD-Tg mice treated with immunebasedvaccination as compared to PBS-treated controls (Student's t-test;P=0.0028). FIG. 4j depicts a decrease in mean plaque area, observed inthe retinas of vaccinated AD-Tg mice as compared to their controls(Student's t-test P=0.0002). FIG. 4k shows that the significantreduction in the total area covered by plaques was also detected in thebrain hippocampus and cortex of the same mice following immune-basedvaccination (Student's t-test P=0.0085). Error bars in each panelrepresent SEM.

FIGS. 5a through 5k depict in vivo imaging of curcumin-labeled plaquesin AD-Tg mouse retinas. FIGS. 5a-5c are representative z-axis projectionimages taken from retinal whole-mounts of non-perfused AD-Tg versusnon-Tg (wt) mice (10-month-old), following i.v. curcumin or PBSadministration in vivo (blood vessels are indicated by red arrows). FIG.5a shows Aβ plaques were visible (indicated by white arrows) in AD-Tgmouse retinas following i.v. injection of curcumin (n=6). FIG. 5b showsplaques were undetectable in the retinas of AD-Tg mice after i.v.injection of PBS (n=5). FIG. 5c shows plaques were undetectable in theretinas of non-Tg (wt) mice following i.v. injection of curcumin (n=5).FIG. 5d is a representative confocal z-axis projection image, usingthree channels and sagittal/coronal virtual sections, demonstrating Aβplaques (plaques inside the vessels indicated with white arrows),stained with anti-human Aβ antibodies, in the parenchyma and inside theblood vessels of AD-Tg mouse retinal whole-mount. FIGS. 5e and 5f depictimages captured using a fluorescence microscope with AOTF-based spectralimaging system, and analyzed and visualized by segmentation andclassification software. In FIG. 5e , Aβ plaques (in white) and bloodvessels (indicated by arrows), were visible in a retinal whole-mountstained in vivo with curcumin and imaged at a single channel (ex. 562/40nm; em. 624/40 nm). FIG. 5f depicts a spectrally-classified image usingoptical signature (OS) specific to Aβ plaques labeled with curcumin inthe same retinal whole-mount and region. Aβ plaques are shown inpseudocolor (indicated by white arrows) and all non-plaque tissue is ingreen pseudocolor. Scale bars=10 μm. FIGS. 5g-5j are images following asingle injection of curcumin, wherein plaques (indicated with whitearrows) were visible in live AD-Tg mouse retinas (n=4) by emission oflight following excitation with a spectrally controlled source(wavelength of 546/15 nm). FIGS. 5i and 5j are higher magnificationimages, where plaques were mostly detected in areas close to the opticdisc and the average plaque size was compatible with that observed inthe whole-mount retinas (ex vivo). FIG. 5k shows no plaques detected inthe non-Tg (wt) mice (n=4) i.v. injected with curcumin. Scale bars (g,k)=100 μm, and (h-j)=10 μm. 23.

FIG. 6 depicts a flow diagram of a spectral imaging system fordiagnosing, prognosing, and analyzing Aβ plaques in accordance with anembodiment of the present invention.

FIG. 7 depicts a flow diagram of a spectral imaging system fordiagnosing, prognosing and/or analyzing Aβ plaques in accordance with anembodiment of the present invention.

FIGS. 8a through 8d depict high-resolution images of small retinalplaques (mostly<1 μm in diameter), which were found to originate fromthe endogenous mouse APP gene. Images are of a 10 month old Non-Tg(wt)mouse retina. FIG. 8a (curcumin), FIG. 8b (mouse Aβ), FIG. 8c (DAPI) andFIG. 8d (Merged).

FIG. 9 depicts images of live AD-Tg (upper right and lower right panels)and Non-Tg(wt) (upper left and lower left panels) mouse retinas showingcurcumin stained plaques in the AD-Tg retina, and the lack of curcuminstained plaques in the Non-Tg(wt) retina.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed, J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5^(th) ed., J.Wiley & Sons (New York, N.Y. 2001); and Sambrook and Russel, MolecularCloning: A Laboratory Manual 3rd ed, Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y. 2001), provide one skilled in the art with ageneral guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

“Administering” and/or “Administer” as used herein refer to any routefor delivering a pharmaceutical composition to a patient. Routes ofdelivery may include non-invasive peroral (through the mouth), topical(skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular andrectal) and inhalation routes, as well as parenteral routes, and othermethods know in the art. Parenteral refers to a route of administrationthat is generally associated with injection, including intraorbital,infusion, intraarterial, intracarotid, intracapsular, intracardiac,intradermal, intramuscular, intraperitoneal, intrapulmonary,intraspinal, intrasternal, intrathecal, intrauterine, intravenous,subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.Via the parenteral route, the compositions may be in the form ofsolutions or suspensions for infusion or for injection, or aslyophilized powders.

“Alzheimer's Disease” as used herein refers to all form of dementia,identified as a degenerative and terminal cognitive disorder. Thedisease may be static, the result of a unique global brain injury, orprogressive, resulting in long-term decline in cognitive function due todamage or disease in the body beyond what might be expected from normalaging.

“Age-related macular degeneration” as used herein refers to is a medicalcondition in older adults that results in a loss of vision in the centerof the visual field (the macula) due to damage to the retina.

“Cataracts” as used herein refers to a clouding that develops in thecrystalline lens of the eye or in its envelope, varying in degree fromslight to complete opacity and obstructing the passage of light. Earlyin the development of age-related cataract the power of the lens may beincreased, causing near-sightedness (myopia), and the gradual yellowingand opacification of the lens may reduce the perception of blue colors.Cataracts typically progress slowly to cause vision loss and arepotentially blinding if untreated.

“Fluorescent Marker” as used herein refers to any and all compoundscontaining fiurophore for attaching the compound to another molecule,such as a protein or nucleic acid. This is generally accomplished usinga reactive derivative of the fluorophore that selectively binds to afunctional group contained in the target molecule.

“Glaucoma” as used herein refers to a group of diseases that affect theoptic nerve and involves a loss of retinal ganglion cells in acharacteristic pattern. Glaucoma is categorized as a type of opticneuropathy.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees, and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats and guineapigs, and the like. The term does not denote a particular age or sex.Thus adult and newborn subjects, as well as fetuses, whether male orfemale, are intended to be including within the scope of this term.

“Therapeutically effective amount” as used herein refers to that amountwhich is capable of achieving beneficial results in a mammal beingtreated. A therapeutically effective amount can be determined on anindividual basis and can be based, at least in part, on consideration ofthe physiological characteristics of the mammal, the type of deliverysystem or therapeutic technique used and the time of administrationrelative to the progression of the disease, disorder or condition beingtreated.

“Treat,” “treating” and “treatment” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) the targeted pathologiccondition, disease or disorder even if the treatment is ultimatelyunsuccessful. Those in need of treatment may include those already withthe disorder as well as those prone to have the disorder or those inwhom the disorder is to be prevented.

β-Amyloid deposition is central to AD neuropathology and a key hallmarkof Alzheimer's disease. However, monitoring Aβ plaques in the brains ofliving Alzheimer's patients and animals is limited by the currentresolution and specificity of MRI and PET, and a definite diagnosis ofAlzheimer's or other ailment or condition characterized by the formationof Aβ plaques is only possible after brain tissue autopsy by monitoringnumber and distribution of plaques and tangles. Hence, developing meansto identify plaques in vivo is essential for diagnosis as well as forevaluation of disease progression in response to therapies.

The present subject matter establishes the formation of retinal Aβplaques in mammals and teaches a method for identifying, quantizing, andimaging retinal Aβ plaque. The present subject matter may beincorporated for patients with Alzheimer's disease, dementia, and otherclinical conditions and ailments characterized by the formation of Aβplaques. Furthermore, the present subject matter discovered that theformation of Aβ plaques in the retina of AD patients preceded theirappearance in the brain. Accordingly, the present subject matterdiscloses a method for early diagnosis of AD in a mammal comprising thesteps of administering a fluorescent marker to the patient for stainingAβ plaques in the retina, and imaging the retina of the patient with aoptical imaging system to identify stained Aβ peptides.

Another embodiment of the present subject matter teaches a method forprognosing AD in mammals by measuring the increase or decrease of Aβplaques in the retina of patients before and after treatment. The methodof prognosis comprises the steps of administering a fluorescent markerto the patient for staining Aβ plaques in the retina and imaging theretina of the patient with an optical imaging system to identify stainedAβ peptides, followed by administering an AD treatment to the patientand allowing due course for the AD treatment to take effect. And,re-administering a fluorescent marker to the patient for staining Aβplaques in the retina after AD treatment and imaging the retina of thepatient with the optical imaging system to identify an increase ordecrease in stained Aβ peptides.

In a further embodiment, the present subject matter discloses a methodfor treating AD in mammalian patients, comprising administering atherapeutically effective amount of myelin-derived peptides and/oragonist of myelin-derived peptides to the patient in reducing theformation of, and dissolving the existence of Aβ plaques.

The present subject matter also finds utility in disclosing methods forimproving eyesight in mammalian patients containing retinal Aβ plaques,comprising the steps of administering a therapeutically effective amountof myelin-derived peptides and/or agonist of myelin-derived peptides tothe patient. The method of improving eyesight may be applicable forpatients with AD, dementia, or other clinical conditions and ailmentscharacterized by the formation of Aβ plaques, such as Age-RelatedMacular Degeneration (AMD), and glaucoma.

In further embodiments the subject matter describes that Aβ plaques arepresent in the retina of mammals and may be utilized to analyze,prognose and diagnose a multitude of other clinical conditions andailments characterized by retinal Aβ plaques. Representative clinicalcondition and ailments may include AMD and glaucoma.

Further discoveries identified in the present subject matter include anoptical imaging system for visualizing Aβ plaques in vivo in the retinaof non-human mammalian and human patients. The optical imaging systemincorporates the use of a fluorescence microscope, mercury and xenon arclamps, a CCD camera, an AOTF (acousto-optic tunable filters)-basedspectral image acquisition apparatus, and post-analysis imagingsoftware. The optical imaging system incorporates the foregoing tools toprovide retinal images of stained Aβ plaques, providing a visualpseudo-color representation of the spectral signature extracted from theraw images, representing the size and location of the Aβ plaquesobjects.

In an alternative embodiment, the optical imaging system incorporatesthe use of a stereomicroscope that is adjusted to visualize fluorescenceand scatter signals at higher resolutions. The stereomicroscope may befitted with a Polychrome V variable wavelength light source. Inadditional embodiments, the optical imaging system may incorporate aMicroFire color digital camera and one or more magnifying lenses toimprove magnification and image detail. Image acquisition is attainedand perfected by post-analysis image segmentation and classificationusing imaging software.

In further embodiments, the optical imaging system may be incorporatedin methods for diagnosing, prognosing, and treating Aβ plaques inmammals. Furthermore, the optical imaging system may be augmented withadaptive optics, used to improve the performance of the optical imagingsystem by reducing the effects of rapidly changing optical distortion.

In yet another embodiment, the subject matter method may be utilized fordrug development and testing. As the non-invasive, rapidly repetitiveimaging methods would enable back-to-back comparison of various drugsand various dosage of drugs, the present subject matter would findfavorable utility in drug development and testing.

A previous report has identified Aβ pathology in the brain, based on thefinding of Aβ accumulation in the lenses of AD patients [31]. Thecurrent study provides evidence for the existence of Aβ plaques in theretinas of AD patients that could be specifically visualized bycurcumin. plaques were found in the retina of all examined AD patients,whereas they could not be detected in the non-AD controls. In both youngas well as in aged AD mice, a good correlation between retinal and brainAβ plaque pathology was observed; plaques accumulated in anage-dependent manner during disease progression, and both retina andbrain showed Aβ plaque reduction as a response to the same therapeuticmodality. Overall, the retinal tissue, which shares many similaritieswith the brain, can potentially be used for diagnosis and monitoring ofAD.

In the present study, Aβ plaques in the retinas of the AD patients weredetected mostly within the RGC layer. In AD mouse eyes, plaques wereseen in most of the retinal layers and in the choroid. Plaques werenoticeable from the NFL to the ONL, and clusters of Aβ plaques were seenmore often in the inner layers of the retina, signifying the possibilityof plaque imaging through the eyes of living subjects. Retinas of ADmice undergo an age-dependent increase in Aβ plaque load in terms ofboth number and size, similar to the age-dependent accumulation ofplaques observed in the brain. Our results demonstrating retinal plaquepathology are consistent with a recent report that reveals retinal Aβdeposition in correlation with retinal inflammation and degeneration inadult and aged AD-Tg mice [16]. The present subject matter, we not onlyprovides evidence supporting a link between retinal and brain plaquepathology, but also show that Aβ plaques are detectable in the retinaprior to their detection in the brain, in young AD-Tg mice. We werefurther able to show a significant reduction of Aβ plaques in theretinas of AD-Tg mice following vaccination with myelin-derived peptide;this treatment as well as related ones were found to be effective inattenuating Aβ plaque burden in the brain [17, 24, 25]. These findingsprovide that assessment of retinal plaques may be used to evaluateresponses to plaque-reducing therapy, and that retinal plaques mayrespond to the same treatment that is effective in Aβ plaque reductionin the brain.

Importantly, the fact that plaques were seen in the GCL in the humaneyes, reaching a size of more than 5 makes imaging Alzheimer's patientsthrough the retina a feasible approach, with some modifications, evenwith the currently available tools for human eye imaging, such as anadaptive optics ophthalmoscope [32]. In live mice, a commerciallyavailable mydriatic retinal camera, was found to be effective inrecording fundus photographs enabling evaluation of longitudinal changesof retinal ganglion cells [33]. A blue-light confocal scanning laserophthalmoscope (bCSLO) system that was modified to visualize cyanfluorescent protein, also provides a noninvasive approach to visualizeRGCs in the living mouse retina [34]. Here, for the proof-of-concept, wewere able to detect curcumin-labeled plaques in live mice using astereomicroscope (Leica S6E) equipped with Polychrome V spectral lightsource and double convex lens. Moreover, using an AOTF system, we wereable to detect retinal Aβ plaques by curcumin while eliminating strongbackground autofluorescence signals (from red blood cells).

In the present study, curcumin was effective in detecting retinal Aβplaques when systemically administered at a single dose of 7.5 mg/kg orwhen given orally. Curcumin demonstrated the ability to cross theblood-brain and blood-retina barriers, which is a requirement for auseful plaque-imaging agent. In terms of safety, Phase I and II trialsusing curcumin in patients with cancer have proven its low toxicity inhumans even at high doses (12 g/day), and when given over extendedperiods of time [35]. Translation of curcumin doses given intravenouslyor orally, from mice to humans (below 1 g) for retinal plaquevisualization, is expected to remain within the reported safety levels.Furthermore, recent studies have reported various approaches tosignificantly increase curcumin stability and bioavailability in humans[36].

The identification of Aβ plaques in the retina of AD patients, providesa novel opportunity for developing a high resolution and sensitiveimaging method, that will allow their detection in vivo. These resultsmay be consistent with the early visual dysfunctions found in ADpatients [37,38], and with the evidence for retinal abnormalities suchas loss of cells in the GCL and atrophy of the NFL, reported in ADpatients [39-44]. Although it is unclear whether Aβ plaques are found inthe retina at early or later stages of AD, the current discovery of Aβplaques in the retina of these patients at different ages, and the factthat these plaques are detectable at a very early pre-symptomatic phaseof the disease in AD-Tg mice, strengthens the possibility thatcurcumin-labeled plaques, seen through the eyes, could be used for earlydiagnosis of AD. Importantly, based on their unique size anddistribution within the retinas, the plaques observed in AD patientscould be eventually used for differential diagnosis: plaques that weredetected in age-related macular degeneration are locally restricted toretinal pigment epithelium within drusen and appear smaller in size[45-47]. In terms of the retinal abnormalities seen in AD patients, itis possible that a plaque-reducing therapy, such as the current DCvaccination, may also help to ameliorate some of the visualdysfunctions, even leading to improved eyesight.

Along with aging of the world's population and the growing epidemic ofAD, an early detection of AD becomes ever more critical for evaluatingrisk, assessing new therapies, and treating AD with early interventiononce it has developed. AD pathology, including amyloid plaques andneurofibrillary tangles, is believed to appear many years beforesymptoms manifest and before any substantial neurodegeneration occurs.Discovery of early measurable markers specific to AD, such as theAβ-plaques in the retina, which may predict development of brainpathology and cognitive decline in still cognitively normal subjects, isespecially needed. The inventors' findings in mice models of AD supportthe use of imaging of retinal plaques in vivo labeled with curcumin as anon-invasive tool for early indication of AD pathology and response to atherapeutic intervention.

Furthermore, the present subject matter introduces vaccination therapiesto reduce and/or eliminate Aβ-plaques in the retina, often associatedwith degeneration of the eyes and eyesight in AD patients.Myelin-derived peptides or weak agonists of myelin-derived peptides wereused to effectively induce neuroprotection and to reduce plaqueformation in the retina.

In summary, we identified Aβ plaques in human retinas, and describe anew approach to detect and monitor Alzheimer's plaque pathology earlierand more readily than in the brain, by imaging Aβ plaques in the retinausing a systemically administered compound, proven safe in humans. Thismay predict development of brain pathology and cognitive decline insubjects who are still cognitively normal and well before a significantfunctional deficit is seen. These findings show that optical imaging ofthe retina can be used as a noninvasive approach for monitoring ADprogression and response to therapeutic interventions [48].

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Results Aβ Deposits in AD Mouse Retina can be Visualized UsingCurcumin

AD-Tg mice carrying the human APPswe and PS1dE9 transgenes were used toassess the potential of developing a noninvasive tool for detecting Aβplaques in the eye. We first verified that curcumin had an affinity tothe same plaques that were detected by antibodies specific to human Aβin the hippocampus of AD-Tg mice (FIG. 1a ; separate channels FIGS. 1band 1c ). At higher magnification, images show the specific stainingpattern obtained following each procedure (FIGS. 1d and 1e ). HumanAβ-plaques were undetectable in the brains of non-Tg littermate wildtype (wt) mice (FIG. 1f ). We then tested whether Aβ plaques in the eyesof AD-Tg mice could also bind curcumin. Examination at high resolutionrevealed the presence of Aβ plaques labeled by both curcumin andanti-human AP-antibodies in the retinas of AD-Tg mice (FIG. 1g retinalwhole mount; FIG. 1k-1m , cross-section) but not in the retinas ofnon-Tg (wt) mice (FIGS. 1j and 1n ). The representative images displaythe location of Aβ plaques in retinal whole mounts at various depths(consecutive acquisition at focal planes of 80 μm depth) to include theinner plexiform layer (IPL; FIG. 1g ), inner nuclear layer (INL)/outerplexiform layer (OPL; FIG. 1h ), and outer nuclear layer (ONL; FIG. 1i). Analysis of cross sections further verified Aβ plaque deposition indeep retinal layers and the choroid, with an apparent predominance inthe ganglion cell layer (GCL) and IPL through OPL layers (FIGS. 1k-1m ).Whereas human-Aβ plaques were absent in the non-Tg (wt) littermates(FIGS. 1j and 1n ), occasional small curcumin-positive plaques weredetected. To determine the nature of these small and sparse plaques thatwere detected by curcumin staining in wt mice, we carried out adouble-staining experiment using curcumin and antibodies specific tomouse-Aβ in wholemount retinas of 10-month old wt mice. Indeed, thesmall plaques detected by curcumin in the wt retinas were found to beco-labeled with the anti-mouse Aβ antibodies, thus confirming theiridentity as endogenously formed mouse-Aβ deposits (FIGS. 8a-8d ).

Example 2 Results Aβ Plaques are Formed in Retinas of AD Patients andcould be Visualized by Curcumin

We next examined the presence of Aβ plaques in postmortem eyes ofpatients with definite diagnosis of AD (n=9; age range from 48 to 94years; different disease severities, categorized based on theirneuropathology reports), and in postmortem eyes of age-matched normalcontrols (n=4; 66 to 92 years; see human donor eye records in (see Table1). Autofluorescence and non-specific signals of fixed human eyesobserved under excitation ranging from 360-710 nm and associated withlipofuscin/lipid deposits and/or long-term fixation with formalin [21,22], were eliminated by Sudan Black B staining (FIGS. 2a-2j ). Forcurcumin staining, we first immersed human whole-mount retinas withSudan Black B (FIGS. 2a and 2c ; no plaques were observed), followed byexposure to curcumin (FIGS. 2b and 2d ; representative images displayplaques within the same tissue location). Plaques detected by curcumin,ranging in size from 1 to 10 μm (typically around 5 μm), were found inall AD patient eyes examined, at various focal depths corresponding tothe GCL, IPL and INL retinal layers (FIGS. 2a and 2g ), and with anapparent correlation to the reported plaque pathology in the brain. Wealso analyzed the human retinas with antibodies directed against humanAβ. We identified Aβ plaques in AD patients and found that theirstructure was similar to that found in the mouse retina and brain [FIGS.2h and 2i represent the innermost retinal layers (i.e. GCL) where theplaques are easily detected; FIG. 2i is a higher magnification image ofthe retinal Aβ plaque structure; FIG. 2j represents deeper consecutivefocal planes (i.e. IPL)]. The plaques could not be detected when onlysecondary antibodies were used (data not shown). Exposure of the humanretinas to curcumin after their immunolabeling for Aβ, confirmed theirco-localization (FIGS. 2k-2m ). In non-AD human eyes, no Aβ plaques weredetected (FIG. 2n ).

TABLE 1 Patient Gender¹ & Pre-Mortem Diagnosis Post-Mortem Final # Age(yrs) (Disease Duration) Neuropathology Diagnosis Cause of Death 412 F,48 Dementia, AD Moderate to frequent NPs AD Cerebral atrophy (10 yrs)and NFTs in the neocortex definite with and hippocampus hydrocephalus404 F, 65 Dementia, AD Large no. of diffuse AD N/A (5 yrs) plaques, NPs,NFTs in the definite entorhinal cortex and hippocampus 435 M, 70Dementia, AD Moderate no. of NPs and AD Pneumonia of (5 yrs) NFTsdefinite posterior lungs 539 M, 78 Dementia Large no. of NPs most ADSubdural (3 yrs) with cores and abundant definite hematoma NFTs anddiffuse plaques 484 M, 86 Dementia, AD Abundant NFTs and NPs AD Cerebrum& (11 yrs) in the neocortex and definite Cerebellum hippocampusinfarction 664 M, 87 Dementia, AD Moderate to frequent NPs AD Cerebrum(8 yrs) with NFTs in the definite infraction neocortex and HippocampusCA-1 486 F, 88 Dementia, AD Severe NPs and NFTs AD Pneumonia (6 yrs)definite 513 F, 90 Dementia, AD² Mild NPs³ and abundant AD Cerebral (14yrs) NFTs⁴ definite⁶ hemorrhage infarction focal 525 F, 94 Dementia, ADAbundant NPs with AD Cerebrum (11 yrs) mature cores and definiteinfarction numerous NDTs 93-78  M, 66 Absence of N/A Normal Liverfailure Dementia Brain Normal 93-111 M, 77 Absence of N/A Normal SepsisDementia Brain Normal 476 M, 88 Absence of Sparse neocortical NPs NormalCerebrum Dementia and NFTs Brain infarctions Occipital CVA⁵ 529 F, 92Absence of Moderate no. of diffuse Normal Cerebrum Dementia plaques andsmall no. of Brain infarction remote Normal NPs only in the multiplehippocampus ¹Gender: F = Female. M = Male ²AD—Alzheimer's Disease³Neuritic Plaques (NPs) and ⁴Neurofibrillary Tangeles (NFTs) weredetermined by Silver (Galiyas or Bielschowsky) strain and Thioflavinstain in several CNS Sites: Hippocampus CA-1. Entorhinal Cortex. MidFrontal. Sup./Mid. Temporal. Inferior parietal. Primary Visual. Visualassociation area. ⁵CVA—Cerebral vascular accident or stroke. ⁶ADdefinite—According to CERAD criteria.

Example 3 Results Aβ Plaques in AD Mice, Stained In Vivo by Curcumin,are Detected in the Retina Earlier than in the Brain and AccumulateDuring Disease Progression

To establish the use of curcumin for imaging plaques in the retina, wetested its bioavailability to the eye when injected systemically. Tothis end, mice were intravenously injected with curcumin. Labeledplaques following the administered curcumin could be detected in theretinas and brains of AD-Tg mice, but not in the non-Tg (wt) controls(FIGS. 3a-3q ). These findings confirmed that curcumin crosses theblood-brain barrier and suggest that it also crosses the blood-retinabarrier and has a high affinity for Aβ plaques in vivo. Importantly,curcumin-labeled plaques could be detected following a single curcumininjection or following multiple injections. Representative z-axisprojections of retinal and brain hippocampus and cortical images ofAD-Tg mice at the ages of 2.5, 5, 9 and 17 months demonstrated anage-dependent correlation between plaque deposition in the retina andthe brain, and increased accumulation over the course of diseaseprogression (FIGS. 3a-3n ). Importantly, plaques were detected in theretina (FIGS. 3a and 3b ) but not in the brain (FIGS. 3c and 3d ) asearly as at 2.5 months of age in AD-Tg mice following in vivo curcuminadministration, suggesting that Aβ plaques in the retinas precede brainpathology. We further confirmed that these curcumin-labeled plaques wereco-localized ex vivo with anti-human Aβ antibody staining (FIGS. 3b and3f ). Aβ plaques were first detectable in the brain at the age of 5months (FIGS. 3g and 3h ), in line with previous descriptions of diseaseinitiation and progression in this strain of AD-Tg mice [23]. In the wtmice, Aβ plaques were undetectable both in the retina (FIG. 3o ) and inthe brain (FIGS. 3p and 3q ) as late as 9 months of age.

Example 4 Results Aβ Plaque Burden is Decreased in the Retina FollowingVaccination Therapy

We further investigated whether the fate of retinal plaques observed inAD-Tg mice, is similar to that of brain Aβ plaques in response to thesame treatment. Myelin-derived peptides or weak agonists ofmyelin-derived peptides have been shown to effectively induceneuroprotection and to reduce plaque formation [24-26]. To ensure thebeneficial effect of the vaccination without the risk of inducingautoimmune encephalomyelitis, we chose to vaccinate AD-Tg mice with analtered myelin-derived peptide (MOG45D, derived from MOG 35-5527, 28)using dendritic cells (DCs) as a carrier and adjuvant. Whole-mountedretinas of 10-month old AD-Tg mice injected with either MOG45D-loadedDCs or with PBS, and those of wt littermates (4 mice/8 retinas pergroup), were ex vivo labeled for Aβ plaques, using both curcumin andanti-Aβ antibody (FIGS. 4a-4k ). Representative axial (z-stack)projection images demonstrated substantial reduction of the number of Aβplaques in vaccinated AD-Tg mice compared to PBS-treated controls (FIGS.4a-4c versus FIGS. 4d and 4f , respectively; separate channels in FIGS.4b and 4c , and FIGS. 4e and 4f ). No Aβ plaques (double stained withcurcumin and antihuman Aβ antibody) were detected in the wt mice (FIG.4g ). In high-resolution images, we occasionally detected small retinalplaques (mostly<1 μm in diameter), which were found to originate fromthe endogenous mouse APP gene (FIGS. 8a-8d ). These small plaques werestained by curcumin but not by anti-human Aβ antibodies in all threeexperimental groups (FIGS. 4a, 4d and 4g ). We further quantified plaquenumber and size by capturing 12 areas (total of approx. 0.45 mm²) aroundthe optic disc, and quantified plaques across a 60-μm scanning depth ineach area (FIG. 4h ; each area is indicated by rectangle 1-12). Asignificant decrease in plaque number detected by curcumin staining wasfound in the retinas of vaccinated AD-Tg mice compared to PBS-treatedcontrols (FIG. 4i ; P=0.0028). Substantial reduction was also observedin the average area covered by the retinal plaques in vaccinated versusPBS-treated AD-Tg mice (FIG. 3j ; P=0.0002). Notably, significantreduction, relative to PBS-treated mice, in the total plaque area wasalso observed in the hippocampus and cortex from the brains of the samevaccinated mice (FIG. 4k ; P=0.0085).

Example 5 Results In Vivo Imaging of Aβ Plaques in the Eyes UsingSystemically Injected Curcumin

To further investigate the potential of visualizing Aβ plaques bycurcumin in the eyes of live subjects, we first tested our ability toidentify Aβ plaques in whole-mount retinas of mice that were notperfused prior to their eye enucleation, a more physiological setting.Representative axial projection images demonstrated that even underthese conditions, which included background signals from red blood cellsin the capillaries, plaques could be identified in the retinas of AD-Tgmice that had been previously i.v. injected with curcumin (FIG. 5a ).Importantly, in the absence of curcumin, plaques were undetectable inAD-Tg mice that had been i.v. injected with PBS (FIG. 5b ), suggestingthat when using these imaging modalities, plaques are barely detectablein the retina solely by their autofluorescence signals. As expected, innon-Tg(wt) mice injected with curcumin, plaques were also not detected(FIG. 5c ). Additional labeling of plaques with anti-human Aβ antibodiesex vivo, confirmed the Aβ specificity of curcumin staining (data notshown). Aβ plaques in whole-mount retinas of ADTg mice labeled withanti-Aβ antibodies were found inside blood vessels as well as in theirparenchymal vicinities (FIG. 5d ; confocal virtual cross-section). Wefurther assessed whether it would be possible to detect Aβ plaques whilereducing the background signal emerging from blood vessels. To this end,we monitored the specific optical signature using a fluorescencemicroscope (Nikon TE2000) including a multi-spectral imaging technology,comprised of spectral imaging with acousto-optic tunable filters (AOTF)[29] and fluorescence lifetime imaging using a gated camera; imageacquisition was followed by post-analysis image segmentation andclassification using software that was previously developed by us [30].Curcumin-labeled plaques imaged using a microscope equipped with AOTF ata single wavelength channel were observed in AD-Tg mouse retina (FIG. 5e). By applying the AOTF-based imaging, capturing the spectral signatureof curcumin-labeled plaques, and post-translation into color-classifieddigital images, we were able to identify the specific optical signatureof Aβ plaques as “true” signals, while eliminating the autofluorescencenoise generated by the blood vessels (FIG. 5f ). To investigate thefeasibility of our approach for noninvasive plaque detection, weconducted an in vivo imaging of the retina in live mice using a modifiedstereomicroscope (Leica S6E) with a wavelength-controlled light sourceand a digital camera. Following a single injection of curcumin (7.5mg/kg) two hours prior to imaging, curcumin-labeled plaques were visiblein AD-Tg mice retina specifically at an excitation wavelength of 546/15nm (FIGS. 5g-5j ). Plaques were mostly detected in areas close to theoptic disc. The average plaque size was compatible with that observed inthe whole-mount retina (ex vivo). No plaques were detected in the non-Tg(wt) mice injected i.v. with curcumin (FIG. 5k ) or in AD-Tg mice thatdid not receive curcumin injection (data not shown). To verify that thesignals captured by the modified stereomicroscope originated from theplaques, mice were euthanized, and the presence of the curcumin-labeledplaques was confirmed on whole-mount retinas (data not shown).

Example 6 Mice

Double-transgenic mice (females and males at equal numbers) that harborthe chimeric mouse/human APP (APPswe) and the mutant human presenilin 1(deletion in exon 9 PSEN1.DELTA.E9) genes and their aged-matched non-Tglittermates, were purchased from the Jackson Laboratories (Bar Harbour,Me., strain #4462) and were bred and maintained in the animal center ofcomparative medicine of Cedars-Sinai Medical Center (Los Angeles,Calif.). All experiments were approved and conducted according toregulations devised by the Cedars-Sinai Institutional Animal Care andUse Committee.

Example 7 Genotyping

Genomic DNA was extracted from 0.5 cm tail tip using a DNA extractionkit (Qiagen, Valencia, Calif.) following the manufacturer's protocol.Mice used in this study were genotyped for the presence of thetransgenes by PCR as previously described (Jankowsky, 2004, Ref #120).

Example 8 Vaccination Preparations

Modified myelin-derived peptide (MOG45D) is derived from theencephalitogenic peptide MOG₍₃₅₋₅₅₎ (Koehler, 2002, Ref #285; Shao,2002, Ref. #283; Zhong, 2002, Ref. #284; Hauben, 2001, Ref. #28; andHauben, 2001, Ref. #35). For vaccinations, MOG45D (Invitrogen, Carlsbad,Calif.) was added to bone marrow-derived dendritic cells from non-Tglittermates' donor mice. Preparation of dendritic cells for vaccinationwas as previously described (Hauben, 2003, Ref. #34).

Example 9 Experimental Regimen for Vaccinations

AD-Tg mice at the age of 7 months were injected with DC-MOG45 (0.5×10⁶cells/200 ml in 1×PBS per animal) once a month, for three months.Control groups of 7-month-old AD-Tg mice were injected with 1×PBSaccording to the corresponding regimens. At the end of the study, allmice were perfused under anesthesia with 1×PBS following 2.5%paraformaldehyde (“PFA”) (Sigma) and their brains and eyes werecollected for further analysis.

Example 10 Animal Tissue

Mice were anesthetized and perfused with 4% ice-cold buffered PFA, and agroup of mice were not perfused. Their eyes were enucleated and fixedimmediately in 4% fresh PFA overnight. For whole mount retinas, the eyeswere dissected and the anterior part was removed. The eyecups weresoaked for 10 minutes in hyaluronidase (type I-S) (0.07 mg/ml) (Sigma)to liquefy and remove the vitreous residues, than washed for 10minutes×3 in PBS, and the whole mount retinas were collected. For wholeeye sectioning, the eyes were put in 30% sucrose in 4% PFA for 2 hours,than washed for 15 minutes×3 in PBS. The eyes were embedded in O.C.T andfrozen slowly on dry ice than sagittal sectioned (7 μm) with cryostat.Brains were collected and fixed immediately in 4% fresh PFA overnight.The brains were put in 30% sucrose (in 4% PFA) gradient. Brains werewashed for 15 minutes×3 in PBS, next embedded in O.C.T and frozen slowlyon dry ice, then coronal sectioned (30 μm) with cryostat.

Example 11 Human Autopsy Eyes

Autopsy eyes from Alzheimer's patients were procured from theAlzheimer's Disease Research Center, Department of Pathology, Universityof Southern California (Los Angeles, Calif.), under IRB protocols 99491and 3201. Healthy donor eyes were purchased from National DiseaseResearch Interchange (NDRI, Philadelphia, Pa.). NDRI has a human tissuecollection protocol approved by a managerial committee and subject toNational Institutes of Health oversight. Diseased and normal eyes werefixed and stored in 10% neutral buffered formalin. In addition, we usedtwo healthy eyes that were frozen without fixation and stored at −80° C.Whole-mount retinas were prepared from the eyes and further studied byimmunohistochemisty.

Example 12 Tail Vein Injection of Curcumin

For in vivo Aβ-plaque imaging, AD-Tg and non-Tg wild-type mice wereintravenously injected to the tail vein with curcumin in PBS (7.5mg/kg/day, for 7 consecutive days) or with PBS. Subsequently, brains andeyes were cryosectioned, or prepared for whole mount retina. In analternative embodiment, curcumin may be administered to the patientorally.

Example 13 Immunohistochemistry

Brain cryosections (30 μm thick), retina cross sections (cryosections)(7 μm thick) and whole mount retinas were treated with apermeabilization/blocking solution containing 20% horse serum(Invitrogen) and 0.01-0.1% Triton X-100 (Sigma-Aldrich, St. Louis, Mo.).Sections were stained overnight at 4° C. with a specified combination ofthe following primary Abs in PBS containing 10% blocking solution: mouseanti-Aβ [human amino-acid residues 1-17; clone 6E10 (1:100; Milipore,Temecula, Calif.)]. The sections were incubated for 1 hour in roomtemperature with secondary Abs, then washed three times with 1×PBS andmounted using Vectorshield containing or not4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, VectorLaboratories, Peterborough, UK). Secondary Abs solution in 1×PBScontained Cy-5-conjugated donkey anti-mouse antibody (1:200; JacksonImmunoResearch Laboratories, West Grove, Pa.). A negative control wasprocessed with the same protocol with the omission of the primaryantibody to assess nonspecific labeling. For microscopic analysis weused a Zeiss ApoTome fluorescent.

Example 14 Curcumin Staining

Curcumin solution at 0.1 mg/ml was prepared by dissolving curcumin(Sigma-Aldrich) in 0.5M NaOH, pH=7.9, following immediate dilution in1×PBS. Brain and retina tissue cryosections (30 μm and 7 μm thickrespectively) and whole mount retinas were stained with curcuminsolution for 10 minutes in room temperature, then rinsed three timeswith 1×PBS for 15 minutes each. The samples were covered with GVAmounting media (Zymed).

Additional compounds are known in the art that can stain/label in vivoamyloid plaques, including, Thioflavin S and T and some derivatives,Congo Red and derivatives, methoxy-X04, Pittsburgh Compound-B (PiB),DDNP, Chrysamine-G and several more. However, curcumin and it'sderivatives are very appealing for in vivo optical imaging of amyloidplaques in animal models as well as humans, because of the followingadvantage. Curcumin generates specific and very bright signals in thecommonly used optical spectrum, and is commercially available,exceptionally low cost. Safety issues related to curcumin are minimal(even at high dosages) and may even consider to be beneficial to thepatient's health as an antioxidant. Curcumin is an effective ligand withvery good in vitro and in vivo binding characteristics to Aβ, and offersgood initial brain uptake and washout rate from the brain (importantproperties for in vivo imaging agents).

Example 15 Quantification

Micrographs of stained tissues were obtained on an Axio Imager Z1ApoTome-equipped microscope (with motorized Z-drive) with AxioCam MRmmonochrome camera ver. 3.0 (at a resolution of 1388×1040 pixels, 6.45μm×6.45 μm pixel size, dynamic range of >1:2200, that delivers low-noiseimages due to Peltier-cooled sensor). Quantitative analysis of Aβ plaquenumber and area (mm²) was performed from two wholemounted retinas permouse (n=4 mice per group). Each image, captured at 40× objective withresolution of 0.28 μm, included an area of 0.04 mm², and a total of 12rectangular areas around the optic disc within scanning depth of 60 μm(multiple virtual section images at consecutive focal planes using amotorized scanning stage). Measurements of the average plaque radius(following curcumin staining) were completed for each animal groupfollowed by calculation of the average plaque area in each animal group.For the acquisition, we used similar exposure times (approx. 75 ms) andthe same gain values (0) for all images. No image post-processing waspreformed. The emission signals of Aβplaques stained with curcumin werecompared to the background signals in the retinal tissue, to determinesignal to background ratio. The calculated signal-to-background noiseratio from the images was high and within the range of 3:1 to 10:1.Quantitative analysis of Aβ plaque number and area (mm²) in the brainwas determined from three coronal sections (two hemispheres each) permouse with 450 μm intervals, over an area covering hippocampal andcortical regions. Optical sections from each field of the specimen wereimported into the NIH Image J program (National Institutes of Health).Conversion to greyscale was performed to distinguish between areas ofimmunoreactivity and background. Total area and quantitative levels ofimmunoreactivity were determined using a standardized customhistogram-based threshold technique, and then subjected to particleanalysis.

Example 16 Spectral and Multispectral Imaging

Spectral imaging provides digital images of an object at a large,sequential number of wavelengths generating precise optical signaturesat every pixel. The fluorescence spectral signature of Aβ plaques,labeled in vivo with curcumin, was captured by our spectral imagingsystem using the following equipment: Nikon fluorescence microscopes(E800 and TE2000), mercury and xenon arc lamps, a CCD camera, an AOTF(acousto-optic tunable filters)-based spectral image acquisition system(ChromoDynamics, Inc)[29] and post-analysis imaging software developedby our Minimally Invasive Surgical Technologies Institute [30]. Thefinal images provided a visual pseudo-color representation of thespectral signature extracted from the raw images, representing the sizeand location of the analyzed objects. In multispectral imaging,fluorescence lifetime imaging, performed with a pulsed laser and aLaVision PicoStar HR gated camera, was supplementing the spectralacquisition.

FIG. 6 depicts a flow diagram of a spectral imaging system 100 fordiagnosing, prognosing, and analyzing Aβ plaques in vivo in accordancewith an embodiment of the present invention. The subject matter retina110 is stained with a fluorescent marker to label Aβ plaques. Shortlythereafter, the stained retina 110 is imaged by an imaging device 120that is adjusted to visualize fluorescence and scatter signals at higherresolutions. The imaging device 120 may be fitted with a Polychrome Vvariable spectral light source 130. In additional embodiments, thespectral imaging system 100 may incorporate a color digital camera 140(e.g. MicroFire) and one or more magnifying lenses to improvemagnification and image detail. Image acquisition 170 is attained bypost-analysis image segmentation and classification using imagingsoftware 160.

FIG. 7 depicts a flow diagram of a spectral imaging system 200 fordiagnosing, prognosing, and analyzing Aβ plaques in accordance with anembodiment of the present invention. The subject matter retina 210 isstained with a fluorescent marker to label Aβ plaques. Shortlythereafter, the stained retina 210 is imaged using an imaging device220. The imaging device 220 may be fitted with multi-spectral imagingtechnology, comprising of spectral imaging with acousto-optic tunablefilters (AOTF) 230 and fluorescence lifetime imaging using a digitalcamera 240. Image acquisition 260 is attained by post-analysis imagesegmentation and classification using imaging software 250.

Example 17 In-Vivo Imaging of Mouse Retina

AD-Tg and wt mice retinas were imaged two hours following curcuminintravenous injection. Mice were anaesthetized with Ketamine 100mg/ml/kg and Xylazine 20 mg/ml/kg. Mouse pupils were dilated to about 2mm in diameter with 0.5% phenylephrine hydrochloride ophthalmic solution(Bausch & Lomb) combined with 0.5% tropicamide ophthalmic solution(Mydral; Bausch & Lomb). During the imaging process, the mice werepositioned on a stage of the stereomicroscope, and the eye was coveredwith a drop of PBS supplemented with calcium and magnesium, which servedas an optical coupling medium between the eye surface and the imaginglens. A modified stereomicroscope (Leica S6E) that was adjusted tovisualize fluorescence and scatter signals at higher resolution was usedto capture images (exposure time 750 ms. with gain 4). The modifiedstereomicroscope was assembled to include a Polychrome V (TillPhotonics) variable wavelength light source, a MicroFire color digitalcamera (Optronics), and an additional 6× (double convex) magnifyinglens, with a focal length of 10 cm. Images were repeatedly captured atseveral different angles, in order to visualize a larger field and toeliminate non-specific reflection signals.

Example 18 Statistical Analysis

Results were analyzed by one tailed unpaired Student's t test for the pvalues of two-group comparison. Results are expressed as means±SD.

Various embodiments of the present subject matter are described above inthe Detailed Description. While these descriptions directly describe theabove embodiments, it is understood that those skilled in the art mayconceive modifications and/or variations to the specific embodimentsshown and described herein. Any such modifications or variations thatfall within the purview of this description are intended to be includedtherein as well. Unless specifically noted, it is the intention of theinventors that the words and phrases in the specification and claims begiven the ordinary and accustomed meanings to those of ordinary skill inthe applicable art(s).

The foregoing description of various embodiments of the present subjectmatter known to the applicant at this time of filing the application hasbeen presented and is intended for the purposes of illustration anddescription. The present description is not intended to be exhaustivenor limit the subject matter to the precise form disclosed and manymodifications and variations are possible in the light of the aboveteachings. The embodiments described serve to explain the principles ofthe present subject matter and its practical application and to enableothers skilled in the art to utilize the subject matter in variousembodiments and with various modifications as are suited to theparticular use contemplated. Therefore, it is intended that the presentsubject matter not be limited to the particular embodiments disclosedfor carrying out the subject matter.

While particular embodiments of the present subject matter have beenshown and described, it will be obvious to those skilled in the artthat, based upon the teachings herein, changes and modifications may bemade without departing from this subject matter and its broader aspectsand, therefore, the appended claims are to encompass within their scopeall such changes and modifications as are within the true spirit andscope of this subject matter. It will be understood by those within theart that, in general, terms used herein are generally intended as “open”terms (e.g., the term “including” should be interpreted as “includingbut not limited to,” the term “having” should be interpreted as “havingat least,” the term “includes” should be interpreted as “includes but isnot limited to,” etc.).

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1.-28. (canceled)
 29. A method for diagnosing Alzheimer's disease invivo in a human subject, comprising systemically administering curcuminto the subject for staining retinal Aβ plaques; imaging the subject'sretina with an optical imaging system suitable for visualizing retinalAβ plaques of from 1 μm to 10 μm; examining the images for stainedretinal Aβ plaques of from 1 μm to 10 μm; and diagnosing the subject ashaving Alzheimer's disease if stained retinal Aβ plaques of from 1 μm to10 μm are present.
 30. The method of claim 29, wherein stained retinalAβ plaques are present in the GCL, IPL and INL of the subject's retina.31. The method of claim 29, wherein stained retinal Aβ plaques arepresent in the GCL of the subject's retina.
 32. The method of claim 29,wherein the optical imaging system is selected from the group consistingof a spectrometer, a fluorescence microscope, a stereomicroscope, amercury arc lamp, a variable wavelength light source, a xenon arc lamp,a CCD gated camera, a color digital camera, an acoustic-optic tunablefilter-based spectral image acquisition system, adaptive optics, imagingsoftware, and combinations thereof.
 33. The method of claim 29, whereinthe amount of curcumin administered is less than 12.0 grams and greaterthan 7.5 mg.
 34. A method for diagnosing Alzheimer's disease in vivo ina human subject, comprising systemically administering curcumin to thesubject for staining retinal Aβ plaques; imaging the subject's retinawith an optical imaging system suitable for visualizing retinal Aβplaques of more than 5 μm; examining the image for stained retinal APplaques of more than 5 μm; and diagnosing the subject as havingAlzheimer's disease if stained retinal Aβ plaques of more than 5 μm arepresent.
 35. The method of claim 34, wherein stained retinal Aβ plaquesare present in the GCL, IPL and INL of the subject's retina.
 36. Themethod of claim 34, wherein stained retinal Aβ plaques are present inthe GCL of the subject's retina.
 37. The method of claim 34, wherein theoptical imaging system is selected from the group consisting of aspectrometer, a fluorescence microscope, a stereomicroscope, a mercuryarc lamp, a variable wavelength light source, a xenon arc lamp, a CCDgated camera, a color digital camera, an acoustic-optic tunablefilter-based spectral image acquisition system, adaptive optics, imagingsoftware, and combinations thereof.
 38. The method of claim 34, whereinthe amount of curcumin administered is less than 12.0 grams and greaterthan 7.5 mg.
 39. A method for identifying Aβ plaques in a humansubject's retina in vivo, comprising systemically administering curcuminto the subject for staining retinal Aβ plaques; imaging the subject'sretina with an optical imaging system suitable for visualizing retinalAβ plaques of from 1 μm to 10 μm; and examining the image for stainedretinal Aβ plaques of from 1 μm to 10 μm.
 40. The method of claim 39,wherein stained retinal Aβ plaques are present in the GCL, IPL and INLof the subject's retina.
 41. The method of claim 39, wherein stainedretinal Aβ plaques are present in the GCL of the subject's retina. 42.The method of claim 39, wherein the optical imaging system is selectedfrom the group consisting of a spectrometer, a fluorescence microscope,a stereomicroscope, a mercury arc lamp, a variable wavelength lightsource, a xenon arc lamp, a CCD gated camera, a color digital camera, anacoustic-optic tunable filter-based spectral image acquisition system,adaptive optics, imaging software, and combinations thereof.
 43. Themethod of claim 39, wherein the amount of curcumin administered is lessthan 12.0 grams and greater than 7.5 mg.
 44. A method for identifying Aβplaques in a human subject's retina in vivo, comprising systemicallyadministering curcumin to the subject for staining retinal Aβ plaques;imaging the subject's retina with an optical imaging system suitable forvisualizing retinal Aβ plaques of more than 5 μm; and examining theimage for stained retinal Aβ plaques of more than 5 μm.
 45. The methodof claim 44, wherein stained retinal Aβ plaques are present in the GCL,IPL and INL of the subject's retina.
 46. The method of claim 44, whereinstained retinal Aβ plaques are present in the GCL of the subject'sretina.
 47. The method of claim 44, wherein the optical imaging systemis selected from the group consisting of a spectrometer, a fluorescencemicroscope, a stereomicroscope, a mercury arc lamp, a variablewavelength light source, a xenon arc lamp, a CCD gated camera, a colordigital camera, an acoustic-optic tunable filter-based spectral imageacquisition system, adaptive optics, imaging software, and combinationsthereof.
 48. The method of claim 44, wherein the amount of curcuminadministered is less than 12.0 grams and greater than 7.5 mg.